| In this paper, heterologous Lumbrokinase gene F238mature peptide were cloned into Pichia pastoris yeast multiply-copied vector and E.coli.-Bacillus subtilis shuttled vector to express Lumbrokinase in yeast and Bacillus subtilis. The optimum fermentation culture conditions of the engineering strains were confirmed also. The main results were as follows:Heterologous genes which were expressed in yeast and Bacillus subtilis were amplified by PCR using pPIC9-F238as template with different primers. The mature peptide gene F238of Lumbrokinase was amplified and cloned into vector pPIC3.5K and pPBE2R to construct expression plasmid pPIC3.5K-F238and pPBE2R-F238.Study in expression of Lumbrokinase in Pichia pastoris GS115. Expression vector was digested with BglⅡ and transformed into Pichia pastoris GS115. First of all, selected his+Mut5phenotype transformants, then screened strains which could resistance high concentration of G418. In the research, we got a positive recombinant strain. The transformant was induced by methanol, SDS-PAGE suggested the relative molecular weight of heterologous expression productor was about2.8×104Da, in correspondence with the theoretical value. The fibrinolytic activity was detected with fibrin plate method, the fibrinolytic activity was up to257.6U/mL.Study in expression of Lumbrokinase in Bacillus subtilis WB600. Expression vector pPBE2R-F238was transformed into Bacillus subtilis WB600by chemical transformation. SDS-PAGE suggested the relative molecular weight of heterologous expression productor was about2.8×104Da, in correspondence with the theoretical value. The fibrinolytic activity of the engineering strain was up to213.7U/mL. Fermentation medium and fermentation conditions of the engineering strain were optimized. Cultured seed solution at age18h with40mL medium in250mL flask shaking22h at37℃, the final activity reached593.5U/mL. Medium containing20g/L of corn flour,10g/L soybean meal,0.4g/L KCl,0.1g/L MgSO4,0.3g/L CaCl2,0.05g/L FeCl3, and pH is7.0. |
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